Summary This proposal is focused on characterizing the innate antiviral immune response of Drosophila. In particular, the response to infection with a DNA virus, Invertebrate Iridescent Virus 6, will be characterized. In preliminary studies,IIV-6 infection was found to induce the robust expression of a group of JAK-STAT responsive genes known as the Turandots, or Tot, genes. The eight Tot genes encode of a family of rapidly-evolving, small, secreted proteins with no known functions. Viral-induced Tot expression requires viral replication, p38b MAPK signaling, and JAK-STAT activation. Additionally, the Unpaired (UPD) cytokines, which are the only known JAK-STAT ligands, distantly related to mammalian IL-6, are up-regulated after DNA virus infection in a p38b-dependent manner. In vivo, both the p38b and JAK-STAT pathways are required for survival from DNA virus infection. Together, this data suggests that viral infection leads to p38b activation and the subsequent production of the secreted Unpaired cytokines. UPDs, in turn, activate JAK-STAT signaling to induce Tot gene expression and other target genes. In addition, we have found that DNA virus-infected cells produce soluble factors that protect naive cells from viral infection. This antiviral activity interferes with infection of both DNA and RNA viruses. Thus, DNA virus infection induces a broad antiviral state in Drosophila cells. Given the the inducible expression and antimicrobial peptide-like characteristics of the Tots, these ?ndings lead us to hypothesize that virus-induced Turandot proteins function as secreted antivirals. Aim 1 explores the potential of the Tots as antiviral peptides, while Aim 2 proposes an unbiased approach to characterize and identify the soluble antiviral activity induced by DNA virus infection. Altogether, this project will probe the molecular mechanism of Tot gene induction and characterize the DNA virus-induced antiviral state.